Zinc regulates the stability of repetitive minisatellite DNA tracts during stationary phase.

Repetitive minisatellite DNA tracts are stable in mitotic cells but unstable in meiosis, altering in repeat number and repeat composition. As relatively little is known about the factors that influence minisatellite stability, we isolated mutations that destabilize a minisatellite repeat tract in the ADE2 gene of Saccharomyces cerevisiae. One mutant class exhibited a novel color segregation phenotype, "blebbing," characterized by minisatellite instability during stationary phase. Minisatellite tract alterations in blebbing strains consist exclusively of the loss of one 20-bp repeat. Timing experiments suggest that these tract alterations occur only after cells have entered stationary phase. Two complementation groups identified in this screen have mutations in either the high-affinity zinc transporter ZRT1 or its zinc-dependent transcriptional regulator ZAP1. The Deltazrt1 mutant specifically affects the stability of minisatellite tracts; microsatellites or simple insertions in the ADE2 reading frame are not destabilized by loss of ZRT1. The Deltazrt1 blebbing phenotype is partially dependent on a functional RAD50. Zinc is known for its role as an essential cofactor in many DNA-binding proteins. We describe possible models by which zinc can influence minisatellite stability. Our findings directly implicate zinc homeostasis in the maintenance of genomic stability during stationary phase.

Novel checkpoint pathway organization promotes genome stability in stationary-phase yeast cells.

Most DNA alterations occur during DNA replication in the S phase of the cell cycle. However, the majority of eukaryotic cells exist in a nondividing, quiescent state. Little is known about the factors involved in preventing DNA instability within this stationary-phase cell population. Previously, we utilized a unique assay system to identify mutations that increased minisatellite alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here we conducted a modified version of synthetic genetic array analysis to determine if checkpoint signaling components play a role in stabilizing minisatellites in stationary-phase yeast cells. Our results revealed that a subset of checkpoint components, specifically MRC1, CSM3, TOF1, DDC1, RAD17, MEC3, TEL1, MEC1, and RAD53, prevent stationary-phase minisatellite alterations within the quiescent cell subpopulation of stationary-phase cells. Pathway analysis revealed at least three pathways, with MRC1, CSM3, and TOF1 acting in a pathway independent of MEC1 and RAD53. Overall, our data indicate that some well-characterized checkpoint components maintain minisatellite stability in stationary-phase cells but are regulated differently in those cells than in actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element; rather, it may be loss of the checkpoint proteins' other functions that contributes to DNA instability.

Multiple pathways regulate minisatellite stability during stationary phase in yeast.

Alterations in minisatellite DNA repeat tracts in humans have been correlated with a number of serious disorders, including cancer. Despite their importance for human health, the genetic factors that influence minisatellite stability are not well understood. Previously, we identified mutations in the Saccharomyces cerevisiae zinc homeostasis genes ZRT1 and ZAP1 that significantly increase the frequency of minisatellite alteration specifically during stationary phase. In this work, we identified mutants of END3, PKC1, and RAD27 that increase minisatellite instability during stationary phase. Genetic analysis reveals that these genes, along with ZRT1 and ZAP1, comprise multiple pathways regulating minisatellite stability during stationary phase. Minisatellite alterations generated by perturbation of any of these pathways occur via homologous recombination. We present evidence that suggests formation of ssDNA or ssDNA breaks may play a primary role in stationary phase instability. Finally, we examined the roles of these pathways in the stability of a human minisatellite tract associated with the HRAS1 oncogene and found that loss of RAD27, but not END3 or PKC1, destabilizes the HRAS1 minisatellite in stationary phase yeast. This result indicates that the genetic control of stationary phase minisatellite stability is dependent on the sequence composition of the minisatellite itself.

Minisatellite alterations in ZRT1 mutants occur via RAD52-dependent and RAD52-independent mechanisms in quiescent stationary phase yeast cells.

Alterations in minisatellite DNA repeat tracts are associated with a variety of human diseases including Type 1 diabetes, progressive myoclonus epilepsy, and some types of cancer. However, in spite of their role in human health, the factors required for minisatellite alterations are not well understood. We previously identified a stationary phase specific increase in minisatellite instability caused by mutations in the high affinity zinc transporter ZRT1, using a minisatellite inserted into the ADE2 locus in Saccharomyces cerevisiae. Here, we examined ZRT1-mediated minisatellite instability in yeast strains lacking key recombination genes to determine the mechanisms by which these alterations occur. Our analysis revealed that minisatellite alterations in a Δzrt1 mutant occur by a combination of RAD52-dependent and RAD52-independent mechanisms. In this study, plasmid-based experiments demonstrate that ZRT1-mediated minisatellite alterations occur independently of chromosomal context or adenine auxotrophy, and confirmed the stationary phase timing of the events. To further examine the stationary phase specificity of ZRT1-mediated minisatellite alterations, we deleted ETR1 and POR1, genes that were previously shown to differentially affect the viability of quiescent or nonquiescent cells in stationary phase populations. These experiments revealed that minisatellite alterations in Δzrt1 mutants occur exclusively in quiescent stationary phase cells. Finally, we show that loss of ZRT1 stimulates alterations in a derivative of the human HRAS1 minisatellite. We propose that the mechanism of ZRT1-mediated minisatellite instability during quiescence is relevant to human cells, and thus, human disease.